1. Field of the Invention
There is continuing need for simple rapid and accurate qualitative and quantitative determinations of biologically active substances. There is a need for methods that can be conducted by technicians with a low level of skill. In addition there is a need for convenience, reliability and simplicity. In clinical laboratories, there is increasingly a desire for simpler assays that require use of as few reagents and as few steps as possible.
Immunoassays usually employ more than one reagent. In most cases, the reagents cannot be combined in a liquid medium prior to running the assay because they contain components that would react on contact with each other. It is desirable to find a method to combine the active materials in liquid form while preventing the reagents from reacting with each other until such time as a means for releasing one or more of the reagents is provided. Generally in immunoassays the reagents are members of a specific binding pair, consisting of ligand and its complementary receptor, one of which is labelled with a member of a signal producing system. Specific binding pair members that are complementary to each other usually react upon contact. Therefore, such reagents are generally stored separately until just prior to the time an assay is conducted.
One patented technique for combining interreactive agents in a single reagent is to formulate the reagents dry so that no reactions occur until a liquid sample or diluent is added. Dry reagents, however, impose some restraints on assay methods. Achieving a homogeneous blend and avoiding water uptake are matters of concern. Further, premature reaction must be avoided. Dry reagents are expensive and their manufacture and quality control are difficult. For example, it is generally necessary to add the sample and a diluent simultaneously and shake vigorously to assure full dissolution of the powder before the reaction has progressed significantly. Additionally, special processing devices are required.
It is, therefore, desirable to develop a new assay method for determining an analyte in a sample wherein two or more specific binding members are combined in a liquid single reagent. Such a reagent avoids the need for dry reagent blending and shaking and does not require simultaneous addition of sample and diluent. A single liquid reagent decreases the time and skill needed to perform an assay.
2. Description of the Related Art
Litchfield et al., "High Sensitive Immunoassays Based on Use of Liposomes without Complement," Clin Chem 30, 1441-1445 (1984) discuss a liposome-based immunoassay using covalently linked hapten-cytolysin conjugates to lyse vesicles with entrapped enzymes. U.S. Pat. Nos. 3,850,578; 4,483,921; and 4,483,929 disclose immunoreactive liposome reagents in which antigen or antibody is bound to the surface of lipid vesicles. A variety of methods for preparing lipid vesicles are known; see for example, U.S. Pat. Nos. 4,529,561, 4,522,803 and 4,485,054. U.S. Pat. No. 4,311,712 discloses a process for preparing a freeze-dried, liposome mixture.